Quantitative PCR analysis of selected Aspergillus, Penicillium and Paecilomyces species
Quantitative PCR analysis of selected Aspergillus, Penicillium and Paecilomyces species
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DOI：
10.1078/072320204322881826
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发表时间：
2004-03-01
影响因子：
3.4
通讯作者：
Vesper, SJ
中科院分区：
文献类型：
--
作者：
Haugland, RA;Varma, M;Vesper, SJ
开发了总共65种定量PCR（QPCR）测定，其结合了荧光5'核酸酶（TaqMan（R））化学并且针对核核糖体RNA操纵子、内部转录间隔区（ITS 1或ITS 2），并且测试了用于检测选定的曲霉属、青霉属和拟青霉属物种。根据不同生物体中核苷酸序列变异量的不同，试验的特异性从种属或亚种到密切相关的种属组各不相同。还开发并测试了曲霉属、青霉属和拟青霉属所有靶种属的通用测定法。使用以前报道的DNA提取方法，估计的分生孢子检测限为目标物种范围从小于一个到几百个样品的不同测定。在几乎所有情况下，非靶物种的分生孢子检测限至少高出1,000倍。该测定用于分析来自美国各地不同来源的10个HVAC灰尘样品。样品中曲霉属、青霉属和拟青霉属分生孢子的总量（通过通用测定法测定）与特定测定法的总和基本一致，表明样品中所有数量上占优势的菌种均通过特定测定法进行了解释。加标选定靶微生物后对这些样品进行的QPCR分析表明，计数结果在95%的时间内在预期值的约一半对数范围内。有证据表明，通常使用的做法，列举曲霉菌和青霉菌作为一个单一的组或仅属可能会误导了解这些生物体的室内种群及其潜在的健康风险。
A total of 65 quantitative PCR (QPCR) assays, incorporating fluorigenic 5' nuclease (TaqMan(R)) chemistry and directed at the nuclear ribosomal RNA operon, internal transcribed spacer regions (ITS1 or ITS2) was developed and tested for the detection of selected Aspergillus, Penicillium and Paecilomyces species. The assays varied in specificity from species or subspecies to closely related species groups, subject to the amount of nucleotide sequence variation in the different organisms. A generic assay for all target species of Aspergillus, Penicillium and Paecilomyces was also developed and tested. Using a previously reported DNA extraction method, estimated conidia detection limits for target species ranged from less than one to several hundred per sample for the different assays. Conidia detection limits for non-target species were at least 1,000 fold higher in nearly all instances. The assays were used to analyze ten HVAC dust samples from different sources around the US. Total quantities of Aspergillus, Penicillium and Paecilomyces conidia in the samples, determined by the generic assay and the summed totals from the specific assays, were in general agreement, suggesting that all of the numerically dominant species in the samples were accounted for by the specific assays. QPCR analyses of these samples after spiking them with selected target organisms indicated that the enumeration results were within approximately a one-half log range of the expected values 95% of the time. Evidence is provided that the commonly used practices of enumerating Aspergillus and Penicillium as a single group or only by genus can be misleading in understanding the indoor populations of these organisms and their potential health risks.